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Human lung epithelial cells were productively infected with human cytomegalovirus in vitro. Infectious virus was released up to 8 weeks postinfection. The cells retained their morphological characteristics throughout the period of observation, while simultaneously bearing all the features typical of cytomegalovirus infection.
Fetal MRC rat lung primary cell sheets, in which reconstruction of bronchial tissue occurred, were treated with 3-methylcholanthrene (MCA) to examine the carcinogen's biologic effect on lung epithelial elements. Cloned lines were established by subculturing of epithelial outgrowths from primary cell sheets induced by MCA treatment. Newborn MRC rats received an sc injection of these cells to assess their tumorigenicity. These MCA-treated cells at the initial stages of culture temporarily formed benign epithelial structures in scar tissue that developed from an sc injection and then regressed. The epithelial structures were roughly classified as tubular and squamous epithelium. The differentiation of cells in vitro was observed as either a type of lamellar keratinization of the premalignant squamous cell form or a type of cyst formation by premalignant secreting epithelium. With further cultivation, six of 24 cell lines became malignant and, when injected into the animal, induced carcinomas. Qualitative changes in the in vitro differentiation of cells were observed in cell lines that formed squamous cell carcinomas. These changes included spherical keratinization (pearl formation) and single-cell keratinization. MCA treatment induced various premalignant alterations in lung epithelial cells, and the subsequent malignancy seemed to stem from these initial premalignant changes.
Coronaviruses (CoV) emerge suddenly from animal reservoirs to cause novel diseases in new hosts. Discovered in 2012, the Middle East respiratory syndrome coronavirus (MERS-CoV) is endemic in camels in the Middle East and is continually causing local outbreaks and epidemics. While all three newly emerging human CoVs from the past 20 years (SARS-CoV, SARS-CoV-2, and MERS-CoV) cause respiratory disease, each CoV has unique host interactions that drive differential pathogeneses. To better understand the virus and host interactions driving lethal MERS-CoV infection, we performed a longitudinal multi-omics analysis of sublethal and lethal MERS-CoV infection in mice. Significant differences were observed in body weight loss, virus titers, and acute lung injury among lethal and sub-lethal virus doses. Virus-induced apoptosis of type I and II alveolar epithelial cells suggests that loss or dysregulation of these key cell populations was a major driver of severe disease. Omics analysis suggested differential pathogenesis was multi-factorial with clear differences among innate and adaptive immune pathways as well as those that regulate lung epithelial homeostasis. Infection of mice lacking functional T and B cells showed that adaptive immunity was important in controlling viral replication but also increased pathogenesis. In summary, we provide a high-resolution host response atlas for MERS-CoV infection and disease severity. Multi-omics studies of viral pathogenesis offer a unique opportunity to not only better understand the molecular mechanisms of disease but also to identify genes and pathways that can be exploited for therapeutic intervention all of which is important for our future pandemic preparedness.IMPORTANCEEmerging coronaviruses like SARS-CoV, SARS-CoV-2, and MERS-CoV cause a range of disease outcomes in humans from an asymptomatic, moderate, and severe respiratory disease that can progress to death but the factors causing these disparate outcomes remain unclear. Understanding host responses to mild and life-threatening infections provides insight into virus-host networks within and across organ systems that contribute to disease outcomes. We used multi-omics approaches to comprehensively define the host response to moderate and severe MERS-CoV infection. Severe respiratory disease was associated with dysregulation of the immune response. Key lung epithelial cell populations that are essential for lung function get infected and die. Mice lacking key immune cell populations experienced greater virus replication but decreased disease severity implicating the immune system in both protective and pathogenic roles in response to MERS-CoV. These data could be utilized to design new therapeutic strategies targeting specific pathways that contribute to severe disease.
Type II lung epithelial cells were isolated from rabbit lungs using the method of Kikkawa and Yoneda (Kikkawa Y, Yoneda K: Lab Invest 30: 76, 1974). Successful primary cultures were obtained only after utilizing high density cell plating (greater than 3 X 10(5) viable cells per sq. cm.) and allowing an attachment time of 48 hours. Attachment efficiency of the isolated cell preparations was highest when medium was supplemented with 10% fetal calf serum. These conditions enabled us to obtain consistently successful primary type II lung cell cultures. Cultures were monitored for a period of 2 weeks after initiation. Light, phase, and electron microscopy examination demonstrated that these primary cultures were indeed type II cells. The principal morphologic feature was the presence of dense lamellar granules in these cells. These primary cultures retained the characteristic type II features for 3 to 5 days in vitro, after which cultures exhibited a progressive deterioration and loss of their phenotypic properties. This behavioral pattern of type II cells in culture may represent both accelerated proliferation and accelerated transformation of these cells into type I epithelial cells.
Prophylactic inhalation of the synergistic agents ODN M362 and Pam2CSK4 ("Pam2ODN") protects mice against allergic lung disease, including allergic inflammation caused by house dust mite (HDM). By preventing sensitization, Pam2ODN reduces HDM-induced eosinophilic and lymphocytic inflammation. How Pam2ODN affects interactions among lung epithelial cells, dendritic cells, and T cells to prevent eosinophilic lung inflammation remains unclear. In the present study, we show that a single inhaled dose of Pam2ODN before HDM sensitization reduces airway Th2 polarization without affecting Th1 or Treg responses. Furthermore, Pam2ODN pretreatment inhibits the recruitment of lung monocyte-derived dendritic cells (moDCs) and conventional Type 2 dendritic cells (DC2s), while preventing the HDM-induced decrease in conventional Type 1 dendritic cells (DC1s). Bulk RNA-seq of the whole lung reveals that Pam2ODN pretreatment restricts the expression of proinflammatory transcripts induced by HDM sensitization. This tolerogenic effect is also reflected at the single-cell level in lung epithelial cells, where proinflammatory transcripts, pathways, and chromatin accessibility are inhibited. These results indicate that Pam2ODN reprograms lung epithelial cells to attenuate allergen-induced Th2-promoting cytokines and DCs while maintaining the population of protective DC1s. These findings suggest a strategy to mitigate chronic allergic lung diseases.
An epithelial cell line, designated JS-15,4, has been established in culture from jaagiekte lesions and subcultured in vitro for almost 2 years. It exhibits morphological and other features of transformed cells and has been shown by electron microscopy to consist of type B ovine alveolar epithelial cells. Jaagiekte was successfully transmitted to 3 new-born lambs by the intratracheal injection of cells following immunosuppressive treatment with either anti-thymocyte immunoglobulin alone or combined with anti-macrophage immunoglobulin. Incubation periods as short as 10 weeks were recorded. Evidence was also obtained that natural transmission may result from the inhalation of viable cells.
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The ongoing COVID-19 pandemic has led to the emergence of new SARS-CoV-2 variants as a result of continued host-virus interaction and viral genome mutations. These variants have been associated with varying levels of transmissibility and disease severity. We investigated the phenotypic profiles of six SARS-CoV-2 variants (WT, D614G, Alpha, Beta, Delta, and Omicron) in Calu-3 cells, a human lung epithelial cell line. In our model demonstrated that all variants, except for Omicron, had higher efficiency in virus entry compared to the wild-type. The Delta variant had the greatest phenotypic advantage in terms of early infection kinetics and marked syncytia formation, which could facilitate cell-to-cell spreading, while the Omicron variant displayed slower replication and fewer syncytia formation. We also identified the Delta variant as the strongest inducer of inflammatory biomarkers, including pro-inflammatory cytokines/chemokines (IP-10/CXCL10, TNF-α, and IL-6), anti-inflammatory cytokine (IL-1RA), and growth factors (FGF-2 and VEGF-A), while these inflammatory mediators were not significantly elevated with Omicron infection. These findings are consistent with the observations that there was a generally more pronounced inflammatory response and angiogenesis activity within the lungs of COVID-19 patients as well as more severe symptoms and higher mortality rate during the Delta wave, as compared to less severe symptoms and lower mortality observed during the current Omicron wave in Thailand. Our findings suggest that early infectivity kinetics, enhanced syncytia formation, and specific inflammatory mediator production may serve as predictive indicators for the virulence potential of future SARS-CoV-2 variants.
Clones of epithelial-like cells were established from urethan-induced mouse lung adenoma. Electron microscopy of one clone showed that the cells contained lamellar inclusion bodies similar in appearance to those seen in the adenoma precursor, the type II alveolar pneumocyte. The clones exhibited characteristics associated with both "transformed" and "normal" cells in culture; i.e., although aneuploid, the cells grew at a slower rate than most transformed cells, did not form colonies in soft agar and, after prolonged subculture, were not tumorigenic when transplanted s.c. into appropriate hosts. Hydrocortisone treatment of the cloned cells led to growth stimulation and the eventual acquisition of neoplastic potential. Epithelial tumors were produced more readily in athymic, nude mice than in antilymphocyte serum-treated A/He mice. The cells are producing a C-type RNA virus into the culture medium.
Cigarette smoke (CS) exposure is a major risk factor for chronic obstructive pulmonary disease (COPD) and is closely associated with cellular senescence. Previous studies have demonstrated the efficacy of luteolin in treating aging-related symptoms. This study aims to elucidate the therapeutic potential of luteolin against CS-induced cellular senescence. Using a CS-exposed mouse model and cigarette smoke extract (CSE) treated mouse lung epithelial cells (TC-1), we demonstrate that luteolin significantly attenuates CS-induced histopathological alterations and inflammatory cytokine release while alleviating cellular senescence. Transcriptome sequencing suggests that NQO1 and Fos may serve as a common molecular target for both CS-induced pathology and luteolin treatment. Subsequent Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) enrichment analysis and pathway validation experiments revealed that the cAMP agonist Forskolin inhibits senescence marker expression by activating the CREB pathway, exhibiting a mechanism similar to that of luteolin. Notably, luteolin activation of this pathway may not depend on PKA activation. Ultimately, the study found that luteolin mitigates inflammatory responses and prevents lung epithelial cell senescence via the CREB/c-Fos/NQO1 pathway. These findings not only suggest the pivotal role of NQO1 in regulating CS-induced cellular senescence but also underscore the potential of luteolin as a therapeutic drug.
In vivo genome correction holds promise for generating durable disease cures; yet, effective stem cell editing remains challenging. In this work, we demonstrate that optimized lung-targeting lipid nanoparticles (LNPs) enable high levels of genome editing in stem cells, yielding durable responses. Intravenously administered gene-editing LNPs in activatable tdTomato mice achieved >70% lung stem cell editing, sustaining tdTomato expression in >80% of lung epithelial cells for 660 days. Addressing cystic fibrosis (CF), NG-ABE8e messenger RNA (mRNA)-sgR553X LNPs mediated >95% cystic fibrosis transmembrane conductance regulator (CFTR) DNA correction, restored CFTR function in primary patient-derived bronchial epithelial cells equivalent to Trikafta for F508del, corrected intestinal organoids and corrected R553X nonsense mutations in 50% of lung stem cells in CF mice. These findings introduce LNP-enabled tissue stem cell editing for disease-modifying genome correction.
Hypervirulent community-associated MRSA clones such as Staphylococcus aureus (S. aureus) USA300 drive rapidly progressive necrotizing pneumonia with high morbidity and limited therapeutic options. Bacteriophage K (phage K) is a well-characterized lytic phage active against S. aureus, but its efficacy is limited by restricted host range and the emergence of bacterial resistance. Here, we subjected phage K to experimental evolution on S. aureus USA300 to select an adapted variant with enhanced bactericidal properties. Wild-type phage K and the evolved derivative, designated phage KJ25, were compared using growth inhibition assays, time-kill kinetics, genomic differences and transcriptomic analyses of the bacterial response to infection. Efficacy was evaluated in an in vitro A549 lung epithelial cell infection model and ex vivo murine precision-cut lung slices (PCLS). Phage KJ25 exhibited significantly improved killing of USA300, achieving faster bacterial reduction and sustained suppression of regrowth. Genomic analysis identified a function-impairing mutation in gene gp102, encoding a predicted DNA-binding protein implicated in transcriptional regulation. RNA sequencing revealed that KJ25 infection of USA300 induced a slower and less disruptive host transcriptional takeover than wild-type phage K. Importantly, in both A549 cells and PCLS model, phage KJ25 markedly reduced bacterial burden while preserving lung tissue integrity, supporting its therapeutic potential. Collectively, these findings highlight the value of experimental evolution for tailoring therapeutic phages and support phage adaptation as a promising strategy for developing interventions against multidrug-resistant S. aureus.
The distribution of ribonucleoprotein (RNP) within the mitotic spindle of newt lung epithelial cells was studied with the high voltage electron microscope (HVEM) using Bernhard's uranyl-EDTA-lead staining of thick sections in conjunction with the ribonuclease digestion of fixed cells. The results indicate that aside from ribosomes, the major RNP-containing components of the spindle are the kinetochores and centrioles, both of which stain electron-opaque after EDTA treatment. In both cases, the electron-opaque material associated with these microtubule organizing centers (MTOC's) can be removed by RNAse digestion and cold perchloric acid (PCA) extraction under conditions which leave the spindle microtubules (Mts) centrioles, and kinetochores intact. The staining reaction is not abolished by cold PCA extraction alone or by substituting other positively charged proteins (i.e., cytochrome c or lysozyme) for RNAse. The RNP component of the kinetochore is closely associated with the bases of the kinetochore microtubules. The RNP component of the centriole can be seen to surround the microtubules of the triplet blades. No evidence was found to indicate the presence of RNP in the pericentriolar material. The possible function of both kinetochore and centriolar RNP is discussed.
The effect of elevated temperature shocks (ETS) on anaphase chromosome movements and spindle structure was studied in lung epithelial cells cultured from the newt Taricha granulosa granulosa. Mitosis proceeds normally up to temperatures of 31--32 degrees C. At slightly higher temperatures (33--34 degrees C) anaphase is desynchronized while still higher temperatures (35--36 degrees C) arrest chromosome movements. The desynchronization of chromosome movement is related on the ultrastructural level to the re-arrangement of kinetochore fibre microtubules (Mts) into hexagonally packed structures, and with the disappearance of non-kinetochore Mts. During desynchronized movement the distance a chromosome travels depends on the structure of its kinetochore fibre: those which have divergent kinetochore fibre Mts approach the poles, while those with hexagonally packed kinetochore fibre Mts do not. The data provide evidence concerning the anchorage of Mts and demonstrate that normal anaphase chromosome movements are functionally related both to the rearrangement of kinetochore Mts and to Mt disassembly.
BACKGROUND: Streptococcus pneumoniae serotype 3 is one of the most prevalent serotypes that cause invasive pneumococcal disease (IPD) in children and adults worldwide. Serotype 3 is associated with vaccine failures and breakthrough episodes in children vaccinated with 13-valent pneumococcal conjugate vaccine (PCV13). In this study, we aimed to investigate potential genetic mechanisms that could explain the increase in cases of serotype 3 IPD in Spain. METHODS: We analysed the epidemiology of serotype 3 causing IPD in Spain, during 2009-23, in different age groups. Molecular characterisation was performed by whole-genome sequencing. Host-pathogen interactions of the different lineages were evaluated in terms of interaction with lung epithelial cells, biofilm formation, and capsular polysaccharide production, using opsonophagocytosis assays and mouse models of pneumonia. We used Poisson regression models to compare incidence and chi-square test calculations to identify clonal variations across vaccine periods. FINDINGS: Genomic analyses confirmed the predominance of clade I-α/clonal complex (CC) 180 in Spain, which shows increased resistance to complement-mediated immunity and phagocytosis and enhanced potential to infect lung cells. In all isolates of this lineage, we observed a single amino acid substitution (166His→Tyr) in the crucial virulence factor LytA, which increased its enzymatic activity. On evaluating the phagocytosis of mutants without LytA of the two major lineages of serotype 3 (CC180 and CC260), LytA was responsible for the increased phagocytosis-evasion pattern of clade I-α/CC180. Evaluation of individuals with IPD caused by different serotype 3 genotypes confirmed a significant (p<0·05) association between cardiac and respiratory comorbidities and infection by sequence type 180/CC180, which showed the importance of CC180 in IPD. INTERPRETATION: Our findings confirm that PCV13 reduced IPD cases caused by the susceptible CC260 lineage until CC260 was replaced by the clade I-α/CC180 lineage, which has a higher potential to cause IPD and divert the host immune system. A key mutation on LytA protein was associated with this hypervirulent phenotype. Emerging lineages jeopardise the effectiveness of pneumococcal conjugate vaccines. FUNDING: Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III, and PubMLST.
Two groups of weanling pigs, injected with 45Ca, were fed diets containing optimal calcium and phosphorus, and vitamin D3 at 1320 IU/kg feed in the control group, and 825,000 IU/kg feed in the test group. The groups were further subdivided with 2 pigs in each subgroup, with survival times of 1, 2, 3, 4, 7, and 14 days. Pigs fed the high level of vitamin D3 lost weight and anorexia, weakness, rough hair coat and labored breathing were observed. Hypercalcemia began at 12 hours and progressed rapidly after 2 days. Radioisotope sutdies interpreted in the light of histopathologic findings indicated that bone was the primary source of increased plasma calcium. Calcium was released at a rapid rate into blood from prelabeled bone which was undergoing necrosis; it was also removed from blood and deposited into bone at a slower rate due to decreased apposition. Histopathologic examination of bones from test pigs showed regressive changes in the osteocytes, chondrocytes and osteoblasts which bean within 1 day of treatment and resulted in evidence osteopenia within 7 days. Arrested osteocytic osteolysis led to the appearance of cementing lines and to chondroid core retention. Further regressive changes in the osteocytes resulted in osteocytic death and osteonecrosis with subsequent osteoclasia and osteopenia. Retardation and arrest of cartilage maturation as well as osteoblastic deficiency contributed to the osteopenia. The osteopenia was further evidenced by decreased specific gravity and ash content per unit volume of humerus. The initial negative effect on the osteocytes, chondrocytes and osteoblasts is attributed to a direct toxic effect of excessive dietary vitamin D3 since hypoparathyroidism and hypercalcitoninism, which occur secondarily to hypercalcemia, could not account for the rapid appearance of this effect, nor are they known to induce osteocytic death. The release of bone calcium and the resulting hypercalcemia in vitamin D3 toxicosis is therefore due to a direct toxic effect of the vitamin, or its metabolites, on the osteocyte resulting in osteonecrosis. It is not due to increased resorption as has been reported previously from both in vivo and in vitro investigations. Degeneration, with subsequent inflammation, but without calcification, was observed in the kidneys and in the lungs. Epithelial cells, basement membranes, and smooth muscle were affected. This conclusively demonstrates that degeneration is the primary soft tissue lesion in vitamin D3 toxicosis, and that the subsequent calcification is therefore dystrophic. Degenerative changes occurred in the parathyroid glands within 1 day of treatment resulting in necrosis, inflammation and atrophy within 4 days. Relative fibrosis was seen as the parenchyma receded. The parathyroid gland changes were considered a direct effect of vitamin D3 toxicity since they occurred with only mild hypercalcemia and since necrosis of parathyroid cells has not been demonstrated with hypercalcemia either in vivo or in vitro.
Chronic airway diseases such as cystic fibrosis (CF) and primary ciliary dyskinesia (PCD) pose substantial clinical challenges. Here, we explore the p.C97W variant in WAP four-disulfide core domain protein 2 (WFDC2), proposed as a new genetic origin of respiratory distress, especially among Koreans. Whole-exome and whole-genome sequencing (WES/WGS) are performed on 64 patients from 62 families presenting with severe bronchiectasis and chronic rhinosinusitis. Pathogenic variants are found in 19.4% of families, including a novel homozygous WFDC2 missense variant (c.291 C > G, p.Cys97Trp) in five unrelated families. WFDC2 is expressed in lung epithelial cells, and the p.C97W variant impairs WFDC2 protein folding, secretion, and function. Wfdc2 p.C147W knock-in mice exhibit respiratory failure due to the hyperactive epithelial sodium channel (ENaC) linked to increased PRSS8 activity and recapitulate human disease. Treatment with amiloride, an ENaC inhibitor, improves survival and respiratory function in these mice. In conclusion, the p.C97W variant in WFDC2 is a critical genetic factor in severe chronic airway disease that shares clinical features with CF and PCD. Given its implications for diagnosis and treatment, genetic testing for WFDC2 mutations in individuals with CF- or PCD-like symptoms is recommended.
The clinical spectrum of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection ranges from asymptomatic cases to critical COVID-19 pneumonia. To investigate the role of host genetics in susceptibility to critical COVID-19 and identify pathophysiological mechanisms and pathways, we analyzed whole-exome and whole-genome sequencing data from the COVID Human Genetic Effort. We identified 10 rare, monoallelic predicted loss-of-function variants in 18 patients in POLR3A and POLR3C encoding two subunits of RNA polymerase III (POL III), a nuclear multisubunit enzyme, which has been implicated in cytosolic DNA sensing. These variants were deleterious for expression of full-length POLR3A and POLR3C proteins. We demonstrate that human pulmonary A549-hACE2 cells with reduced POLR3A or POLR3C expression exhibit impaired type I IFN responses to transfected mitochondrial DNA (mtDNA) or SARS-CoV-2 infection, together with increased viral replication. Mechanistically, we show that SARS-CoV-2 induces cellular mtDNA release via oligomerization of the mitochondrial voltage-dependent anion channel under virus-induced oxidative stress, enabling POL III-mtDNA interaction. These findings establish POL III as a sensor of endogenous mtDNA released during viral infection and indicate that autosomal dominant POL III haploinsufficiency may predispose individuals to critical COVID-19.